Pawan K. Agrawal

Comparative Genomics and Association Mapping Approaches for Blast Resistant Genes in Finger Millet Using SSRs

The major limiting factor for production and productivity of finger millet crop is blast disease caused by Magnaporthe grisea. Since, the genome sequence information available in finger millet crop is scarce, comparative genomics plays a very important role in identification of genes/QTLs linked to the blast resistance genes using SSR markers. In the present study, a total of 58 genic SSRs were developed for use in genetic analysis of a global collection of 190 finger millet genotypes. The 58 SSRs yielded ninety five scorable alleles and the polymorphism information content varied from 0.186 to 0.677 at an average of 0.385. The gene diversity was in the range of 0.208 to 0.726 with an average of 0.487. Association mapping for blast resistance was done using 104 SSR markers which identified four QTLs for finger blast and one QTL for neck blast resistance. The genomic marker RM262 and genic marker FMBLEST32 were linked to finger blast disease at a P value of 0.007 and explained phenotypic variance (R2) of 10% and 8% respectively. The genomic marker UGEP81 was associated to finger blast at a P value of 0.009 and explained 7.5% of R2. The QTLs for neck blast was associated with the genomic SSR marker UGEP18 at a P value of 0.01, which explained 11% of R2. Three QTLs for blast resistance were found common by using both GLM and MLM approaches. The resistant alleles were found to be present mostly in the exotic genotypes. Among the genotypes of NW Himalayan region of India, VHC3997, VHC3996 and VHC3930 were found highly resistant, which may be effectively used as parents for developing blast resistant cultivars in the NW Himalayan region of India. The markers linked to the QTLs for blast resistance in the present study can be further used for cloning of the full length gene, fine mapping and their further use in the marker assisted breeding programmes for introgression of blast resistant alleles into locally adapted cultivars.

Genome-wide identification, characterization and expression profile of aquaporin gene family in flax (Linum usitatissimum)

Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax.

The development of a phosphite-mediated fertilization and weed control system for rice

Fertilizers and herbicides are two vital components of modern agriculture. The imminent danger of phosphate reserve depletion and multiple herbicide tolerance casts doubt on agricultural sustainability in the future. Phosphite, a reduced form of phosphorus, has been proposed as an alternative fertilizer and herbicide that would address the above problems to a considerable extent. To assess the suitability of a phosphite-based fertilization and weed control system for rice, we engineered rice plants with a codon-optimized ptxD gene from Pseudomonas stutzeri. Ectopic expression of this gene led to improved root growth, physiology and overall phenotype in addition to normal yield in transgenic plants in the presence of phosphite. Phosphite functioned as a translocative, non-selective, pre- and post-emergent herbicide. Phosphite use as a dual fertilizer and herbicide may mitigate the overuse of phosphorus fertilizers and reduce eutrophication and the development of herbicide resistance, which in turn will improve the sustainability of agriculture.

Phosphite: a novel P fertilizer for weed management and pathogen control

Summary The availability of orthophosphate (Pi) is a key determinant of crop productivity because its accessibility to plants is poor due to its conversion to unavailable forms. Weeds competition for this essential macronutrient further reduces its bio‐availability. To compensate for the low Pi use efficiency and address the weed hazard, excess Pi fertilizers and herbicides are routinely applied, resulting in increased production costs, soil degradation and eutrophication. These outcomes necessitate the identification of a suitable alternate technology that can address the problems associated with the overuse of Pi‐based fertilizers and herbicides in agriculture. The present review focuses on phosphite (Phi) as a novel molecule for its utility as a fertilizer, herbicide, biostimulant and biocide in modern agriculture. The use of Phi‐based fertilization will help to reduce the consumption of Pi fertilizers and facilitate weed and pathogen control using the same molecule, thereby providing significant advantages over current orthophosphate‐based fertilization.

Advances in Maize Transformation Technologies and Development of Transgenic Maize

Maize is the principal grain crop of the world. It is also the crop where genetic engineering has been employed to a great extent to improve its various traits. The ability to transform maize is a crucial step for application of gene technology in maize improvement. There have been constant improvements in the maize transformation technologies over past several years. The choice of genotype and the explant material to initiate transformation and the different types of media to be used in various stages of tissue culture can have significant impact on the outcomes of the transformation efforts. Various methods of gene transfer, like the particle bombardment, protoplast transformation, Agrobacterium-mediated, in planta transformation, etc., have been tried and improved over years. Similarly, various selection systems for retrieval of the transformants have been attempted. The commercial success of maize transformation and transgenic development is unmatched by any other crop so far. Maize transformation with newer gene editing technologies is opening up a fresh dimension in transformation protocols and work-flows. This review captures the various past and recent facets in improvement in maize transformation technologies and attempts to present a comprehensive updated picture of the current state of the art in this area.

Stability Performance of Inductively Coupled Plasma Mass Spectrometry-Phenotyped Kernel Minerals Concentration and Grain Yield in Maize in Different Agro-Climatic Zones.

Deficiency of iron and zinc causes micronutrient malnutrition or hidden hunger, which severely affects ~25% of global population. Genetic biofortification of maize has emerged as cost effective and sustainable approach in addressing malnourishment of iron and zinc deficiency. Therefore, understanding the genetic variation and stability of kernel micronutrients and grain yield of the maize inbreds is a prerequisite in breeding micronutrient-rich high yielding hybrids to alleviate micronutrient malnutrition. We report here, the genetic variability and stability of the kernel micronutrients concentration and grain yield in a set of 50 maize inbred panel selected from the national and the international centres that were raised at six different maize growing regions of India. Phenotyping of kernels using inductively coupled plasma mass spectrometry (ICP-MS) revealed considerable variability for kernel minerals concentration (iron: 18.88 to 47.65 mg kg–1; zinc: 5.41 to 30.85 mg kg–1; manganese: 3.30 to17.73 mg kg–1; copper: 0.53 to 5.48 mg kg–1) and grain yield (826.6 to 5413 kg ha–1). Significant positive correlation was observed between kernel iron and zinc within (r = 0.37 to r = 0.52, p < 0.05) and across locations (r = 0.44, p < 0.01). Variance components of the additive main effects and multiplicative interactions (AMMI) model showed significant genotype and genotype × environment interaction for kernel minerals concentration and grain yield. Most of the variation was contributed by genotype main effect for kernel iron (39.6%), manganese (41.34%) and copper (41.12%), and environment main effects for both kernel zinc (40.5%) and grain yield (37.0%). Genotype main effect plus genotype-by-environment interaction (GGE) biplot identified several mega environments for kernel minerals and grain yield. Comparison of stability parameters revealed AMMI stability value (ASV) as the better representative of the AMMI stability parameters. Dynamic stability parameter GGE distance (GGED) showed strong and positive correlation with both mean kernel concentrations and grain yield. Inbreds (CM-501, SKV-775, HUZM-185) identified from the present investigation will be useful in developing micronutrient-rich as well as stable maize hybrids without compromising grain yield.

Successful deployment of marker assisted selection (MAS) for inbred and hybrid development in long-day onion (Allium cepa L.)

In the present investigation, male sterile and maintainer lines were identified using molecular markers in three long day onion populations. Molecular markers, 5’cob and orfA501 were able to distinguish effectively normal (N) and sterile (S) cytoplasm in all the three populations. The observed frequency of S cytoplasm in VL Piaz 67 (100%),VL Piaz3 (86.4%) and KR1 (90%) was higher than N cytoplasm. Out of the two PCR markers viz., OPT and PsaO used to determine the nuclear fertility restorer locus (Ms locus), OPT was found better than PsaO. Increased frequency of dominant homozygous alleles (87.4%) followed by heterozygous alleles (8.6%) and homozygous recessive alleles (4.0%) was observed. Fertility/sterility of plants were validated by acetocarmine staining of the pollens and correlated with observations made using molecular markers. Out of the three populations studied, only 12 plants (2.15%) happened to be completely male sterile in VL Piaz-3 and KR1 population, whereas there was no male sterile plant observed among the population in VL Piaz 67. Selfing and test crossing of plants having normal (N) cytoplasm led to the development and identification of maintainer lines in VL Piaz3 and KR1 population. This is the first example of deploying DNA markers for identification and purification of male sterility and hybrid development in long day onion in Indian population.

Molecular and chemotypic variability of forskolin in Coleus forskohlii Briq., a high value industrial crop collected from Western Himalayas (India)

C. forskohlii (willd.) Briq. is an industrially viable medicinal crop and is widely exploited for the therapeutic potential of its bioactive metabolite, forskolin. The present investigation aimed to explore the chemotypic variability of forskolin content and existing molecular diversity in the wild population of C. forskohlii from the Western Himalayan region of India. Twelve germplasm(s) from different populations were assessed for molecular fingerprinting (ISSR marker) and densitometeric quantification of forskolin. Two elite germplasms viz. NBC-24 (0.728%) and NBC-16 (0.641%) were obtained as the highest accumulator of forskolin with high genetic variability (92%). The UPGMA hierarchical clustering patterns revealed strong genetic grouping between the individuals corresponding to their geographical ranges. Mantel tests showed positive correlation (r = 0.354, p = 0.003) between molecular and chemical fingerprints that reflects the feasibility of the ISSR markers in analyzing genome information related to forskolin biosynthesis from varied phytogeography. Pearson correlation coefficient (0.102) between forskolin content with altitude gradient also denoted a positive correlation. However, the association of both genetic and chemical fingerprinting data with the geographic distance matrix was apparently negative (r = −0.234, p = 0.054; r = −0.067, p = 0.584) which meant that distance might be a predictor of population differentiation. Our study signifies the utility of metabolic and molecular fingerprints for identification of elite accessions and provides a lead to industry for commercial exploitability of Coleus species including its location specific commercial cultivation.

Complementation of sweet corn mutants: a method for grouping sweet corn genotypes

1Vivekananda Parvatiya Krishi Anusandhand Sansthan (VPKAS), Almora 263601, India 2Present address: Division of Genetics, Indian Agricultural Research Institute, New Delhi 110 012, India 3Present address: Department of Genetics and Plant Breeding, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar 263 145, India 4Present address: Indian Council of Agricultural Research Headquarters, New Delhi 110 012, India

Allele mining for resistance gene analogs (RGAs) in crop plants: A special emphasis on blast resistance in finger millet (Eleusine coracana L.)

Finger millet a nutritionally rich underutilized crop requires more attention of research community. One of the major limitations of finger millet for wider agronomic acceptability is because of its susceptibility to blast fungus Magnaporthe grisea, which is also the causative agent of blast in rice. A large amount of sequence data available in the public domain has facilitated identification and isolation of novel genes for blast resistance and other agronomically important traits. Availability of such large genomic data has made allele mining a viable approach for detecting novel alleles for blast resistance in finger millet. However, very scarce genomic information is available in finger millet, being the major hurdle for such approaches. In the present review, we have summarized different strategic approaches suitable for allele mining of resistance gene analogs (RGAs) in finger millet by utilizing the large sequence data available for rice through comparative genomics. This paves the way for transfer of blast alleles into high yielding, blast susceptible and locally well adapted germplasm through molecular breeding and genetic engineering approaches.